Transport in Gram-negative organisms is initiated by passage of the transported species through a transmembrane beta-barrel in the outer membrane. The transport of iron is particularly important for bacterial growth, and outer membrane iron transporters are therefore major vaccine targets against pathogens such as Neisseria, Haemophilus, and Yersinia. These transporters show high affinity and specificity for Fe(III)-ligand complexes, and require energy derived from the proton motive force across the inner membrane to transport ferric complexes. The required energy is provided by transient interaction with an integral inner membrane protein complex, TonB-ExbB-ExbD, resulting in a protein assembly that spans both the inner and outer membranes, as well as the periplasmic space. During FY06, we published work on the following project:[unreadable] [unreadable] Neisseria meningitidis is the causative agent of bacterial meningitis. This blood-borne pathogen acquires iron from human transferrin (hTf = 80 kDa) through an outer membrane transporter complex, transferrin binding proteins A and B (TbpA = 100 kDa; TbpB = 68-85 kDa). TbpA and TbpB form a discrete complex to bind transferrin synergistically, yet each protein is also capable of binding transferrin on its own. TbpA is a TonB-dependent outer membrane iron transporter. We are interested in learning how this human pathogen can extract iron from transferrin and transport it into the periplasm. [unreadable] [unreadable] We have separately expressed and purified TbpA and TbpB in preparation for studying the triple TbpA-TbpB-hTf complex. This work is summarized in annual reports from earlier years. In order to progress with our goal of determining the crystal structure of the triple complex, we first needed to solve the structure of human serum transferrin, a protein which has eluded structure determination for over 30 years.[unreadable] [unreadable] Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependant process. The binding and release of iron results in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. The structure of human serum transferrin (hTF) lacking iron (apo-hTF) which was independently determined by two methods: (1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7 ? resolution using a MAD phasing strategy, by substituting the nine methionines in hTF with selenomethionine and (2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 ? by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human TF and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 62? and 52? rotations are required to open the N- and C-lobe, respectively. Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. [unreadable] [unreadable] Once the full-length hTF structure was available (reference 3 in publication list), we were able to do a structural comparison of hTF and human lactoferrin (reference 4 in publication list). Questions arise as to how TbpA can distinguish hTF from lactoferrin, since the protein share high sequence similarity and bind iron in similar ways. TpbA, however, only binds (and uses iron from) hTF; it does not even bind other closely related TFs. Some of the factors influencing the recognition are identified as follows: Comparison of the structures of iron-free hTF and LTF has revealed several distinctions that could be important in the differing iron and receptor binding properties of these two proteins. Though hTF and LTF are overall quite similar in sequence and structure, they differ in the structure of their inter-lobe linker (helical in LTF and unstructured in hTF), the presence of a salt bridge between the helical linker of LTF and its C-lobe which is absent in hTF, their pattern of disulfide bonding (inter-subdomain bonding in hTF but not in LTF), the relative orientation of their lobes to one another (the C-lobe of LTF is rotated closer to the N-lobe as compared to hTF), the dilysine trigger and triad residues in hTF which are not present in LTF, the openness of their C-lobes (being more open in hTF), the structure of the C-lobe hinge regions (unstructured in hTF and &#946;-strands in LTF), and their inter-lobe interactions (salt bridge between the C-terminal helix and N-lobe of hTF which is not found in LTF). Analysis of these differences increases our understanding of the divergent functions of these two proteins, as well the necessity for pathogenic bacteria to express independent receptors for two such similar proteins. Crystal structures of the bacterial receptors in complex with their host substrates should provide further insight into these interactions.